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- W2894772264 abstract "Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing." @default.
- W2894772264 created "2018-10-12" @default.
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- W2894772264 date "2018-10-02" @default.
- W2894772264 modified "2023-09-26" @default.
- W2894772264 title "Nuts and Bolts of Protein Quantification by Online Trypsin Digestion Coupled LC-MS/MS Analysis" @default.
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- W2894772264 doi "https://doi.org/10.1007/978-1-4939-8814-3_19" @default.
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